DESCRIPTION (Applicant's abstract): To investigate the effect of lung afferent signals on central respiratory rhythm generating circuits, an in vitroneonate rat brainstem/spinal cord preparation, retaining the lungs and their vagal innervation is used. In this preparation, essential rhythm generating circuits are retained, and can be modulated by selective activation of lung slowly adapting mechanoreceptors (SAR). In response to physiological pressures, this in vitropreparation replicates critical aspects of SAR-mediated frequency modulation obtained in vivo: 1) Lung inflation during expiration (E) prolongs E and shifts the phase of respiratory rhythm (Breuer-Hering expiratory reflex; BHE). 2) Lung inflation during inspiration (I) shortens I (Breuer-Hering inspiratory reflex; BHI). 3) SAR feedback increases respiratory frequency when lungs are inflated within 1500 ms of I onset. We will: 1) Identify essential rhythmogenic circuits using SAR modulation as a probe. The BHE is blocked by GABAA receptor antagonist bicuculline, indicating that CV-mediated inhibition is necessary for the reflex. Because the respiratory rhythm is reset, neurons hyperpolarized during mid-expiratory inflation include rhythmogenic neurons. Neurons excited, unmodulated or weakly hyperpolarized during inflation-induced expiratory lengthening are relay neurons, or neurons providing modulatory input to rhythmogenic circuits. This is a critical functional probe to classify respiratory neurons, which based on their firing patterns and intrinsic properties, have resisted simple, consistent classification. 2) Characterize the cellular and synaptic mechanisms for respiratory frequency modulation. Depending on the stimulus phase, inflation can increase or decrease respiratory frequency. In addition, response to SAR afferent input has both a fast and a slow component. Consistent with these observations is the hypothesis that expiratory lengthening is due to hyperpolarization via a paucisynaptic pathway of a subset of rhythmogenic inspiratory neurons, while the increase in frequency accompanying phasic inflation is due to a reorganization of the respiratory network. We propose experiments to test both components of this hypothesis. 3) Describe how sensory feedback transforms the network properties of medullary circuits. At the network level, reincorporating appropriate sensory feedback dramatically increases respiratory frequency. At the single neuron level, firing patterns of respiratory-modulated neurons are transformed. The observed changes shed light on how rhythmogenic circuits in vitro may function in the intact animal.